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ORIGINAL RESEARCH |
Gene Region and Preeclampsia
From the Departments of Clinical Genetics and Human Genetics (AMAL, GP, LPtK), Obstetrics and Gynecology (AMAL), and the Laboratory of Gastrointestinal Immunogenetics (JBAC), Vrije Universiteit Medical Center, Amsterdam, The Netherlands; Department of Obstetrics and Gynecology, North West Adelaide Health Service, University of Adelaide, Elizabeth Vale, Australia (GAD); and University of Iceland, Reykjaví, Iceland (RA).
Address reprint requests to: Augusta M.A. Lachmeijer, MD, Department of Clinical Genetics and Human Genetics, Vrije Universiteit Medical Center, Medical Faculty, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands; E-mail: ama.lachmeijer.humgen{at}med.vu.nl.
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ABSTRACT |
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or lymphotoxin (LT) and preeclampsia.
METHODS: Four di-allelic polymorphisms and five microsatellite markers in the genes encoding TNF- (TNF) and LT (LTA) and their haplotypes were studied in 150 Dutch families. These families contained sib-pairs of women affected with preeclampsia; eclampsia; the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome (strict criteria); or pregnancy-induced hypertension (mild criteria). Frequencies were compared with 98 healthy controls. Nonparametric affected sib-pair analyses for allele sharing among siblings were carried out for all nine markers. Each sibship was composed of an affected index woman and one or more affected sisters.
RESULTS: Although we found a striking association with the TNF-I haplotype in 30 index women with (pre-)eclampsia or HELLP syndrome compared with controls (odds ratio [OR] 3.8; 95% confidence interval [CI] 1.6, 8.9), this association was not found in their 30 sisters meeting similar disease criteria. Analyses in all 150 families showed a similar TNF-I association in 122 index women meeting the strict criteria compared with controls (OR 1.9; 95% CI 1.1, 3.3), but, again, not in their 91 sisters meeting similar disease criteria. This association was stronger in a subgroup of 75 index women with preeclampsia only (OR 2.3; 95% CI 1.2, 4.2). No excess allele sharing for any marker was seen between the siblings.
CONCLUSION: The nine polymorphisms studied in the TNF-LTA region did not show evidence for association or linkage with familial preeclampsia.
Despite evidence for a familial predisposition to preeclampsia,1–3 it has been difficult to identify the genes involved. Genome scans can identify susceptibility loci,4 but studying specific candidate genes could be useful as well. Although the pathogenesis of preeclampsia is unknown, several features of the disease are well established and suggest certain candidate genes.
Cytokines like tumor necrosis factor (TNF) and lymphotoxin (LT) play a central role in immune and inflammatory responses and are therefore prime candidates for the observed activation of the endothelium.5–7 Indeed, increased production of TNF- has been described in support of this hypothesis.8,9 Furthermore, Faas and colleagues created a preeclampsia-like syndrome in pregnant rats using an ultra–low-dose endotoxin infusion protocol,10 and recently Redman et al11 postulated that pregnancy itself causes universal maternal inflammatory responses that might be undermined in preeclampsia.
The TNF and LTA genes are arranged in tandem within the major histocompatibility complex class III region on the short arm of chromosome 6. So far, two case-control association studies between preeclampsia and a polymorphism at position TNF-308 in the promoter region of TNF have been published. Chen and co-workers found an association between the TNF-T1 allele (TNF-308 allele 1) and preeclampsia,12 but others did not find association for the TNF-T1 or -T2 allele.13 These conflicting results could have been due to small sample sizes in these studies, disease heterogeneity, or population heterogeneity. We designed a controlled study with a larger sample set to test the possible association of these loci with preeclampsia using nine markers within a population of 150 sib-pair families.
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MATERIALS AND METHODS |
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Between June 1995 and October 1997, 150 families of affected sisters and their parents were selected for analysis by genome wide scan and candidate gene studies. Recruitment of 2940 women with a history of hypertension in pregnancy was through three routes (Figure 1
). Affected women meeting the strict criteria (n = 448) were selected from medical records of the Vrije Universiteit Medical Center in Amsterdam and the Academic Hospital in Groningen, The Netherlands. Another 2443 affected women were recruited from obstetric databases of 20 other hospitals in The Netherlands using only the crude search criterion of a diastolic blood pressure of at least 100 mmHg throughout pregnancy. Another 49 affected women were recruited through advertisements. Medical family history questionnaires were sent out to all 2940 women. Response was only required when the family history appeared positive. Medical records were examined for all women who responded that they had at least one affected sister (n = 178). Their sisters’ medical records were examined in a similar manner.
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). The "strict index women group" was composed of 122 index women with either preeclampsia or HELLP syndrome. The "strict sisters group" was composed of 91 unrelated sisters with either preeclampsia or HELLP syndrome. The term "unrelated" means that when more than one sister meeting the strict criteria was available in a family, the first recruited sister meeting the same diagnostic criterion from each family was chosen. The "unrelated preeclamptic group" consisted of 101 unrelated women (either index woman or sister) meeting the criterion for preeclampsia only. The "unrelated HELLP group" consisted of 63 women (either index woman or sister) meeting the criterion for HELLP syndrome only.
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We analyzed two di-allelic polymorphisms at positions -308 and -238 in the promoter region of the TNF gene, two di-allelic polymorphisms in the first intron of the LTA gene (LTA-NcoI and LTA-AspHI), and five microsatellite markers: TNFa, b, c, d, and e. In the Dutch population, the alleles of the four di-allelic polymorphisms are seen in only five combinations or haplotypes.15 Figure 215,16 shows how the separate alleles form the haplotypes. In accordance with international nomenclature, these haplotypes are further referred to as TNF-C, -E, -H, -I, and -P.
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Like the di-allelic polymorphism alleles, the microsatellite marker alleles formed haplotypes (data not shown). The total number of microsatellite haplotypes was considerably larger (n = 52) than the five di-allelic polymorphism haplotypes because the number of alleles of each microsatellite marker was around six instead of two. The microsatellite haplotypes were therefore highly informative and could be compared to and matched with the di-allelic polymorphism haplotypes. Details of the distribution of the microsatellite haplotypes over the di-allelic haplotypes will be described elsewhere (article in preparation).
Data analyses of the frequencies of the di-allelic haplotypes are presented as proportions of individuals who are hetero- or homozygous for a given haplotype (+- or ++ frequencies) and as singular haplotype frequencies. Differences in +- or ++ frequencies between the four study groups and controls were calculated by means of 2x2 contingency table analyses and expressed as odds ratios (OR) using Instat 2.02 (Graphpad Software, San Diego, CA). Differences in allele frequencies of the separate di-allelic polymorphisms between the four study groups and controls were similarly expressed as OR with 95% confidence intervals (CI). Two-sided P values <.05 were considered significant.
This study had 80% power to detect an OR of at least 2.3 for the TNF-I haplotype in the strict index women group (n = 122) compared with controls having a 95% CI, based on known haplotype frequencies in the control group. For all other study groups, OR limits were comparable.
Affected sib-pair analyses20 were performed using the Mapmaker/Sibs program (Whitehead Institute for Biomedical Research in Cambridge, MA).21 All analyses were nonparametric; that is, did not require any assumption on the underlying genetic model. The null-hypothesis of this analysis, assuming no linkage between the disease and tested markers, is that 25% of siblings share zero alleles, 50% share one allele, and 25% share two alleles for each marker. Positive linkage between a marker and disease, irrespective of the mode of inheritance, will be reflected by a deviation from these proportions with an excess of allele sharing. Results are given as the distribution of sharing of 0, 1, or 2 parental alleles identical by descent between siblings and as multipoint lod scores. A lod score is the base 10 logarithm of the ratio between the likelihood that observed excess allele sharing is based on linkage between a marker and the disease, and the likelihood that the excess sharing occurred by chance. A lod score above 3.6 is considered significant for linkage.22 The term "multipoint" means that information of all nine markers was used simultaneously. In sib-pair analyses it is possible to exclude a locus of a specific effect. In our analyses we computed lod scores assuming a locus that contributes to a relative risk for siblings (
s) of 2. A s lod score lower than -2 is considered significant evidence for exclusion of linkage. Analyses were done on three levels. In the first "broad" analysis, all women in the 150 pedigrees having disease status according to either the strict or mild criteria were marked as "affected." In the second "intermediate" analysis, only women satisfying strict criteria were marked as "affected"; the disease status of the mildly affected relatives was marked as "unknown." In the final "narrow" analysis, only the most severely affected women (ie, women with eclampsia, severe preeclampsia, or HELLP syndrome) were marked as "affected" and all other relatives were marked as "unknown."
This study was approved by the Medical Ethical Committee of the Vrije Universiteit Medical Center, Amsterdam. Appropriate informed consent was obtained from all participants.
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RESULTS |
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).
To further explore the association found in the index women, we expanded the di-allelic TNF haplotype analysis to the four study groups from the 150 sib-pair families (Table 2). We tested 122 index women meeting the strict criteria (Table 4, strict index women group). We found that the association with the TNF-I haplotype was reduced, though still significant, compared with controls (OR 1.9; 95% CI 1.1, 3.3). The association in the group of 91 sisters meeting the strict criteria (Table 4, strict sisters group) was again not significant. Analyses in the preeclamptic subgroups of the strict index women (n = 75) and strict sisters group (n = 65) again showed a significant association with TNF-I in the index women (OR 2.3; 95% CI 1.2, 4.2), but not in sisters (Table 4). No haplotype associations were apparent in the HELLP subgroups.
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). Again we found a significant association with TNF-I haplotype in the unrelated preeclamptic group (OR 2.1; 95% CI 1.2, 3.7), but not in the unrelated HELLP group. None of the other di-allelic haplotypes (TNF-C, -E, -H, and -P) or microsatellite haplotypes were significantly associated with either preeclampsia or HELLP syndrome. Similarly, no associations were found with any of the individual alleles of the di-allelic markers (data not shown).
To rule out any phenotypic bias between index women and sisters, phenotype characteristics of the siblings from all 150 families were compared. These were very similar (Table 1). Comparisons of these characteristics in subgroups of women with and without the TNF-I haplotype in the index women and sisters group failed to show differences.
The nonparametric affected sib-pair analyses showed no excess allele sharing for any of the markers (Table 5). A plot is made from the "broad" analysis (Figure 3). Included in the plot are also the lod scores assuming a relative risk for siblings (s) for preeclampsia of 2 for any locus in this area. The latter lod scores were around -2 on all marker positions, excluding linkage in this area.
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DISCUSSION |
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and 4, the only comparable finding between index women and sisters was the stronger association with haplotype TNF-I within the preeclamptic subgroups than in the HELLP subgroups. This association was only significant in the index women group. The impact of this stronger association remains limited because comparing women with or without haplotype TNF-I revealed no differences in disease severity. Our control group consisted of healthy males and females representing Dutch population haplotype frequencies. Because the loci studied here are autosomal, sex has no influence on haplotype frequencies. Considering that the incidence of preeclampsia in pregnant women is around 5%, the maximum contribution of potential preeclampsia-associated haplotypes to the control group is 5%. The chance of masking a possible gene effect using this control group is therefore negligible.
Comparing our results with the study of Chen and colleagues,12 which described a significant association of the TNF-T1 allele of the TNF -308 polymorphism with preeclampsia, we found that all but haplotype TNF-E contained this TNF-T1 allele. All haplotype combinations except those with TNF-E involved would thus contain Chen’s T1-T1 genotype. As shown in this study, no consistent associations were seen. Moreover, the genotype frequencies found by Chen and coworkers in their preeclamptic group (T1-T1: 64%, T1-T2: 29%, and T2-T2: 7.1%) were strikingly similar to the genotype frequencies in our healthy control group (T1-T1: 66%, T1-T2: 30%, and T2-T2: 4%). Louis et al23 studied the TNF -308 gene polymorphism in a group of northern European inflammatory bowel disease patients and controls. The T1 and T2 allele frequencies in the healthy control group in that study are very similar to our control group, as were those in the study by Dizon-Townson et al.13 It is possible that the observed frequencies in the Chen and coworkers’ control group were caused by the relatively low numbers of patients and controls (14 women with preeclampsia, 12 with normal pregnancies, and 15 nonpregnant women). There could also be an ethnic bias; it is not clear from the Chen et al study what the ethnic background of the studied groups was.
In the current study, the absence of a consistent association between preeclampsia and the TNF-I haplotype in affected index women and their sisters suggests that involvement of variations at the TNF-LTA loci in familial preeclampsia is unlikely. This is further underscored by the outcome of the nonparametric multipoint allele-sharing analyses. We conclude that there is no linkage or association of any allele of the TNF -308 polymorphism with preeclampsia, nor of any allele of the other eight polymorphisms studied in the TNF-LTA region, which is in agreement with Dizon-Townson and coworkers.13
Kilpatrick recently published a comprehensive review about influences of human leukocyte antigen (HLA) and TNF genes on the development of preeclampsia and the conflicting results in this field so far.24 He concluded that the influence of HLA, related to TNF production, is presumably only a secondary component of genetic susceptibility to preeclampsia. Other factors seem to be involved. These factors could be maternal and fetal in origin. It is likely that combinations of multiple maternal susceptibility genes could generate the same preeclampsia phenotype. Maternal immune maladaptation towards the presence of paternal antigens,6 shedding into the maternal circulation of syncytiotrophoblast microvilli25 or other fetal factors derived from the placenta, in combination with the maternal milieu, could confound discovery of major maternal susceptibility genes for preeclampsia.
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Footnotes |
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The authors thank Esther B. Bastiaans for genotyping all study groups; M. Asunción García-González for genotyping the control group, Jan G. Aarnoudse, A. Salvador Peña, and Maureen Hoatlin for their critical comments on the manuscript; and Lodewijk A. Sandkuijl for his support in linkage analyses.
Received December 7, 2000. Received in revised form June 1, 2001. Accepted June 7, 2001.
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19. Ferencik S, Lindemann M, Horsthemke B, Grosse-Wilde H. A new restriction fragment length polymorphism of the human TNF-B gene detected by AspHI digest. Eur J Immunogenet 1992;19:425–30.[Medline]
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